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      Whole blood transcriptome changes following controlled human malaria infection in malaria pre-exposed volunteers correlate with parasite prepatent period

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          Abstract

          Malaria continues to be one of mankind’s most devastating diseases despite the many and varied efforts to combat it. Indispensable for malaria elimination and eventual eradication is the development of effective vaccines. Controlled human malaria infection (CHMI) is an invaluable tool for vaccine efficacy assessment and investigation of early immunological and molecular responses against Plasmodium falciparum infection. Here, we investigated gene expression changes following CHMI using RNA-Seq. Peripheral blood samples were collected in Bagamoyo, Tanzania, from ten adults who were injected intradermally (ID) with 2.5x10 4 aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria® PfSPZ Challenge). A total of 2,758 genes were identified as differentially expressed following CHMI. Transcriptional changes were most pronounced on day 5 after inoculation, during the clinically silent liver phase. A secondary analysis, grouping the volunteers according to their prepatent period duration, identified 265 genes whose expression levels were linked to time of blood stage parasitemia detection. Gene modules associated with these 265 genes were linked to regulation of transcription, cell cycle, phosphatidylinositol signaling and erythrocyte development. Our study showed that in malaria pre-exposed volunteers, parasite prepatent period in each individual is linked to magnitude and timing of early gene expression changes after ID CHMI.

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          Independent filtering increases detection power for high-throughput experiments.

          With high-dimensional data, variable-by-variable statistical testing is often used to select variables whose behavior differs across conditions. Such an approach requires adjustment for multiple testing, which can result in low statistical power. A two-stage approach that first filters variables by a criterion independent of the test statistic, and then only tests variables which pass the filter, can provide higher power. We show that use of some filter/test statistics pairs presented in the literature may, however, lead to loss of type I error control. We describe other pairs which avoid this problem. In an application to microarray data, we found that gene-by-gene filtering by overall variance followed by a t-test increased the number of discoveries by 50%. We also show that this particular statistic pair induces a lower bound on fold-change among the set of discoveries. Independent filtering-using filter/test pairs that are independent under the null hypothesis but correlated under the alternative-is a general approach that can substantially increase the efficiency of experiments.
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            Protection against a malaria challenge by sporozoite inoculation.

            An effective vaccine for malaria is urgently needed. Naturally acquired immunity to malaria develops slowly, and induction of protection in humans can be achieved artificially by the inoculation of radiation-attenuated sporozoites by means of more than 1000 infective mosquito bites. We exposed 15 healthy volunteers--with 10 assigned to a vaccine group and 5 assigned to a control group--to bites of mosquitoes once a month for 3 months while they were receiving a prophylactic regimen of chloroquine. The vaccine group was exposed to mosquitoes that were infected with Plasmodium falciparum, and the control group was exposed to mosquitoes that were not infected with the malaria parasite. One month after the discontinuation of chloroquine, protection was assessed by homologous challenge with five mosquitoes infected with P. falciparum. We assessed humoral and cellular responses before vaccination and before the challenge to investigate correlates of protection. All 10 subjects in the vaccine group were protected against a malaria challenge with the infected mosquitoes. In contrast, patent parasitemia (i.e., parasites found in the blood on microscopical examination) developed in all five control subjects. Adverse events were mainly reported by vaccinees after the first immunization and by control subjects after the challenge; no serious adverse events occurred. In this model, we identified the induction of parasite-specific pluripotent effector memory T cells producing interferon-gamma, tumor necrosis factor alpha, and interleukin-2 as a promising immunologic marker of protection. Protection against a homologous malaria challenge can be induced by the inoculation of intact sporozoites. (ClinicalTrials.gov number, NCT00442377.) 2009 Massachusetts Medical Society
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              Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

              A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: Project administrationRole: SoftwareRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: InvestigationRole: MethodologyRole: SoftwareRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: MethodologyRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Resources
                Role: Investigation
                Role: Investigation
                Role: ConceptualizationRole: Funding acquisitionRole: Resources
                Role: Funding acquisitionRole: Resources
                Role: Funding acquisitionRole: Resources
                Role: ConceptualizationRole: MethodologyRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                19 June 2018
                2018
                : 13
                : 6
                : e0199392
                Affiliations
                [1 ] Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland
                [2 ] University of Basel, Basel, Switzerland
                [3 ] Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America
                [4 ] Center for Infectious Disease Research, Seattle, Washington, United States of America
                [5 ] Bagamoyo Research and Training Centre, Ifakara Health Institute, Bagamoyo, Tanzania
                [6 ] Sanaria Inc., Rockville, Maryland, United States of America
                University College London, UNITED KINGDOM
                Author notes

                Competing Interests: Authors KLS and SH are affiliated with the commercial company Sanaria Inc., the manufacturer of PfSPZ Challenge. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0003-0770-1689
                Article
                PONE-D-17-43041
                10.1371/journal.pone.0199392
                6007927
                29920562
                d8c5477f-dc9d-4337-9fe4-5a579de3aec8
                © 2018 Rothen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 January 2018
                : 29 May 2018
                Page count
                Figures: 5, Tables: 0, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: U19AI089986
                Award Recipient :
                This work was supported by the National Institutes of Health - Human Immunology Project Consortium grant (number: U19AI089986) to KS. Authors KLS and SH are affiliated with the commercial company Sanaria Inc., the manufacturer of PfSPZ Challenge. The funders provided support in the form of salaries for authors KLS, SH and KS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Parasitology
                Quantitative Parasitology
                Parasitemia
                Medicine and Health Sciences
                Parasitic Diseases
                Malaria
                Medicine and Health Sciences
                Tropical Diseases
                Malaria
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Malarial Parasites
                Medicine and Health Sciences
                Parasitic Diseases
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Plasmodium
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Sporozoites
                Custom metadata
                The Illumina read and normalized count data generated in this study were released to the Gene Expression Omnibus (GEO) database under accession number GSE97158.

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                Uncategorized

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